A number of publications are cited herein in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Each of these references is incorporated herein by reference in its entirety into the present disclosure, to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.
Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.
Ranges are often expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment.
This disclosure includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
RAF, Proliferative Disorders, and Cancer
Mutations in genes that directly or indirectly control cell growth and differentiation are generally considered to be the main cause of cancer. Malignant tumours develop through a series of stepwise, progressive changes that lead to the loss of growth control characteristic of cancer cells, i.e., continuous unregulated proliferation, the ability to invade surrounding tissues, the ability to metastasize to different organ sites, stimulation of angiogenesis, resistance to apoptosis, the ability to evade the immune system, abnormal metabolic pathways, and local inflammation. Carefully controlled in vitro studies have helped define the factors that characterize the growth of normal and neoplastic cells and have led to the identification of specific proteins that control cell growth and differentiation.
RAF is a key downstream target for the RAS Guanine-nucleotide binding/GTPase proteins and mediates the activation of the MAP kinase cascade consisting of RAF-MEK-ERK. Activated ERK is a kinase that subsequently targets a number of proteins responsible for mediating, amongst other things, the growth, survival, and transcriptional functions of the pathway. These include the transcription factors ELK1, C-JUN, the Ets family (including Ets 1, 2, and 7), and the FOS family. The RAS-RAF-MEK-ERK signal transduction pathway is activated in response to many cell stimuli including growth factors such as EGF, PDGF, KGF-, etc. Because the pathway is a major target for growth factor action, the activity of RAF-MEK-ERK has been found to be up-regulated in many factor-dependent tumours. The observation that about 20% of all tumours have undergone an activating mutation in one of the RAS proteins indicates that the pathway is more broadly important in tumourigenesis.
The RAF oncogene family includes three highly conserved genes termed ARAF, BRAF and CRAF (also called Raf-1). RAF genes encode protein kinases that are thought to play important regulatory roles in signal transduction processes that regulate cell proliferation. RAF genes code for highly conserved serine-threonine-specific protein kinases, which are recruited to the plasma membrane following direct binding to RAS, which is the initiating event in RAF activation. RAF proteins are part of a signal transduction pathway believed to consist of receptor tyrosine kinases, p21 RAS, RAF, Mek1 (ERK activator or MAPKK) kinases and ERK (MAPK) kinases, which ultimately phosphorylate several cellular substrates, including transcription factors. Signalling through this pathway can mediate differentiation, proliferation, or oncogenic transformation in different cellular contexts. Thus, RAF kinases are believed to play a fundamental role in the normal cellular signal transduction pathway, coupling a multitude of growth factors to their net effect, cellular proliferation. Because RAF proteins are direct downstream effectors of RAS protein, therapies directed against RAF kinases are believed to be useful in treatment of RAS-dependent tumours.
The RAF kinases are differentially regulated and expressed. CRAF is expressed in all organs and in all cell lines that have been examined. ARAF and BRAF also appear to be ubiquitous, but are most highly expressed in urogenital and brain tissues, respectively. Because BRAF is highly expressed in neural tissues it was once thought to be limited to these tissues but it has since been found to be more widely expressed. Although all RAF proteins can bind to active RAS, BRAF is most strongly activated by oncogenic RAS.
BRAF is important in cancer, because it is mutated in about half of malignant melanomas and thyroid papillary carcinomas, 30% of low-grade ovarian cancer, 15% of colon cancers, and with very high frequency in hairy cells leukaemia, as well as occurring at lower frequencies in a number of other cancers, totaling 7% of human cancers. See, e.g., http://www.sanger.ac.uk/genetics/CGP/cosmic/. A specific subtype of pancreatic cancers, KRAS2 wild-type medullary carcinoma of the pancreas, presents BRAF mutations in 30% of samples (see, e.g., Calhoun et al., 2003). In contrast, ARAF and CRAF mutations are very rare in human cancer.
TABLE 1Tumour TypeFrequencyCitationFrequency of BRAF Mutations in Different Types of CancersMalignant Melanoma50%Papillary Thyroid Cancer (PTC)Xing, 2013Conventional PTC)45%Follicular-variant PTC15%Tall-cell PTC80-100%    Anaplastic Thyroid Cancer25%Colorectal carcinoma15%Low grade ovarian carcinomas30%Hairy Cells Leukemia100% Arcaini et al., 2012Cholangiocarcinoma15%Nervous system tumours 7%Schindler et al., 2011Pilocytic astrocytomas 9%Ganglioglioma18%Pleomorphic xanthoastrocytoma66%Multiple myeloma 4%Chapman et al., 2011Non-Small Cell Lung Cancer1-3% Medullary carcinoma of the30%pancreasFrequency of BRAF Mutations in Other diseasesHistiocytosisLangerhans cell histiocytosis57%Badalian-Veryet al., 2011Erdheim-Chester disease54%Haroche et al., 2012
Over 100 different mutations have been described in BRAF in cancer, but a single mutation (a glutamic acid (E) substitution for the valine (V) at position 600) accounts for about 80% of total BRAF mutations in cancer. This mutant activates BRAF 500-fold, and allows it to stimulate constitutive ERK and NFkB signalling, stimulating survival and proliferation. Consequently, V600E BRAF can transform cells such as fibroblasts and melanocytes. Inhibition of V600eBRAF in cancer cells inhibits cell proliferation and induces apoptosis in vitro; in vivo, it suppresses tumour cell growth, validating V600EBRAF as a therapeutic target.
Other V600 BRAF mutations identified in melanoma are V600K, V6000 and V600R (see, e.g., Davies et al., 2002; Wan et al., 2004; Long et al., 2011; Rubinstein et al., 2010). A minor sub-group of melanomas were also identified with BRAF mutations in positions other than 600. These non-V600 position BRAF mutants do not necessarily activate BRAF kinase activity directly, but require the presence of CRAF to transactivate their MAPK signaling (see, e.g., Smalley et al., 2009). In such cases, inhibition of RAF activity would remain a beneficial aim in cancer treatment.
Importantly, it has been shown that drugs that inhibit mutant BRAF such as vemurafenib (PLX4032, RG7204, Zelboraf) (see, e.g., Flaherty et al., 2010) and dabrafenib (GSK-2118436) (see, e.g., Falchook et al., 2012) can mediate impressive responses in patients whose tumours express oncogenic BRAF (reviewed in Salama et al., 2013). In particular, vemurafenib has shown promising results in mutant BRAF driven melanoma (see, e.g., Chapman et al., 2011; Sosman et al., 2012). It was approved in 2011 by the USA Food and Drug Administration (FDA) for the treatment of V600E BRAF mutant late stage metastatic or unresectable melanoma, and in 2012 by the European Medicines Agency (EMA) as monotherapy for the treatment of adult patients with any BRAF V600 mutation-positive unresectable or metastatic melanoma. Dabrafenib was approved by FDA and EMA in 2013 for the same indication.
These data validate mutant BRAF as a therapeutic target in melanoma and a potential target for other cancers and proliferative diseases where BRAF is mutated. This and other evidence suggests that inhibition of RAF (e.g., BRAF) activity would be beneficial in the treatment of cancer, and that inhibition of RAF (e.g., BRAF) activity could be particularly beneficial in those cancers containing a constitutively activated BRAF mutation.
Resistance to BRAF Inhibitors
Despite being able to mediate significant clinical responses, most patients treated with vermurafenib and dabrafenib eventually progress on treatment (see e.g., Flaherty et al., 2010; Sosman et al., 2012) due to the acquisition of resistance that can be mediated by several mechanisms (see, e.g., Sullivan et al., 2011). Furthermore, about 30% of patients present with primary resistance and do not respond despite the presence of a BRAF mutation (see, e.g., Chapman et al., 2011).
Mutations in KRAS (G12S, G12V, G12D, G12A, G12C, G13A, G13D) have been suggested as predictive markers for identifying tumours that are not susceptible to mutant BRAF inhibitor treatment (see, e.g., Hatzivassiliou et al., 2011). The Q16K mutation of NRAS confers resistance to BRAF inhibitor vemurafenib (see, e.g., Nazarian et al., 2010). Similarly, resistance to treatment with the BRAF inhibitor dabrafenib is predicted by the Q16K and A146T mutations of the NRAS protein (see, e.g., Greger et al., 2012). Activation of RAS through mutations lead to an increase in RAF dimerisation (formation of heterodimer of CRAF with the BRAF protein and/or CRAF/BRAF homodimer) with increased signalling through the MAPK cascade and increased cell proliferation (see, e.g., Poulikakos et al. 2010).
Up-regulation of (and mutations in) CRAF is another mechanism of resistance seen in resistant melanomas treated with BRAF inhibitors (see, e.g., Heidorn et al., 2010; Montagut et al., 2008; Antony et al. 2013). panRAF inhibitors of multiple RAF isoforms (BRAF and CRAF especially) are likely therefore to have an enhanced effect in RAS-mutated melanomas and other RAS mutated cancers, and to address one key resistance mechanism to selective BRAF inhibitors.
Copy number gain of the BRAFV600E gene is associated with BRAF inhibitor resistance in BRAF-mutant melanoma (see, e.g., Shi et al., 2012) and colorectal carcinoma (see, e.g., Corcoran et al., 2010). Both these models are sensitive to concomitant inhibition of BRAF and MEK, but only amplified BRAF-mutant melanoma is sensitive to MEK inhibitor alone. This mechanism of resistance is likely to be more sensitive to panRAF inhibition than to BRAF-only inhibition.
In some melanomas, resistance to vemurafenib was acquired via the expression of splice variant isoforms of BRAFV600E. A 61 kDa splice variant of BRAFV600E (p61BRAFV600E) lacked exons 4-8 encoding the RAS binding domain, and was resistant to vemurafenib. p61BRAFV600E is constitutively dimerised in the absence of activated RAS. Dimerization of p61BRAFV600E was shown to be critical for mediating BRAF inhibitor resistance (see, e.g., Poulikakos et al., 2011).
BRAF and CRAF gene fusion is an alternative mechanism of MAPK pathway activation. These activating gene fusion products have been identified in prostate cancer, gastric cancer and melanoma (SLC45A3-BRAF and ESRP1-RAF1) (see, e.g., Palanisamy et al., 2010), thyroid cancers (AKAP9-BRAF) (see, e.g., Ciampi et al., 2005) and pediatric astrocytomas (KIAA1549-BRAF) (see, e.g., Sievert et al., 2013). Some of the models expressing RAF fusions (for example SLC45A3-BRAF) are sensitive to BRAF and MEK inhibition; in contrast, the KIAA1549-BRAF model is resistant to PLX4720, but is sensitive to a second generation BRAF inhibitor.
Kinase suppressor of Ras (KSR) is a conserved positive modulator of the RAS-RAF-MEK-ERK pathway. KSR1 Interacts constitutively with MEK and is known to play an important role in co-localizing MEK with RAF at the plasma membrane. KSR1 is involved in the MAPK pathway activation by BRAF inhibitors in RAS-mutant or activated RAS cells (see, e.g., McKay et al., 2011). Two mechanisms of drug activation of the pathway have been proposed. One mechanism involves formation of CRAF-KSR1 dimer, complex formation with MEK and MEK phosphorylation by the KSR1-CRAF dimer (see, e.g., Hu et al., 2011). In the other mechanism, KSR1 dimerises with BRAF, and compete with the BRAF-CRAF heterodimer which is the driver of MEK phosphorylation. It was suggested that RAS activated cells with lower expression of KSR1 will show more paradoxical pathway activation (see, e.g., McKay et al., 2011). In both mechanisms, panRAF inhibitors are likely to reduce pathway activation irrespective of KSR1 level of expression.
Overexpression of receptor tyrosine kinases (RTKs) is another mechanism of resistance to BRAF inhibitors. Overexpression of EGFR (epidermal growth factor receptor) leads to EGFR-mediated MAPK pathway reactivation and resistance to vemurafenib in BRAF-mutant colorectal cancers (see, e.g., Corcoran et al., 2012). In drug-resistant BRAF-mutant melanoma cell lines, EGFR-SFK-STAT3 signalling can mediate resistance to BRAF inhibitors in vitro and in vivo, in melanoma (see, e.g., Girotti et al., 2013). Src Family kinases SFKs play a key role in mediating resistance to BRAF inhibitors in melanoma cells (see, e.g., Girotti et al., 2013; Vergani et al., 2011). Elevated phosphorylation of the SFKs LYN, YES and FYN is observed in the vemurafenib-resistant lines. The growth of resistant cells is sensitive to SFK inhibition: Dasatinib and depletion of SRC and LYN both suppressed invasion of the resistant cells in vitro. Of critical importance, SFK signalling was increased in a tumour from a patient with intrinsic resistance to vemurafenib, and dasatinib inhibited the growth and metastasis of this tumour in mice.
Corcoran et al., 2012, “EGFR-mediated reactivation of MAPK signaling contributes to insensitivity of BRAF-mutant colorectal cancers to RAF inhibition with vemurafenib”, Cancer Discovery, Vol. 2, pp. 227-235.
PDGFR-β was found to be overexpressed and hyperphosphorylated in mutant BRAF cell lines resistant to vemurafenib, and upregulated in several cases of vemurafenib-resistant tumours from patients, suggesting that this mechanism may be clinically relevant (see, e.g., Nazarian et al., 2010). Up-regulation of PDGFR-β may drive resistance by activating other ERK1/2-independent downstream pathways (PI3K, PLCγ).
Mechanistic studies showed IGFR1 signalling to mediate increased PI3K/AKT signaling in cells that acquired BRAF inhibitors resistance and that the resistance could be reversed by treating the cells with the combination of a PI3K and a MEK inhibitor or an IGF1R and a MEK inhibitor (see, e.g., Villanueva et al., 2011). The translational relevance of this finding was confirmed by the observation that 1 out of 5 melanoma specimens from patients failing vemurafenib expressed increased levels of IGFR1 (see, e.g., Villanueva et al., 2011).
Growth factor upregulation by the stroma is a mechanism of resistance. A significant correlation has been shown between stromal cell expression of HGF in patients with BRAF-mutant melanoma and innate resistance to RAF inhibitor treatment (see, e.g., Wilson et al., 2012). cMET and/or their ligands are claimed as predictive markers for identifying tumours that are not susceptible to BRAF inhibitor (see, e.g., Hatzivassiliou et al., 2011; Straussman et al., 2012). Dual inhibition of RAF and either HGF or MET resulted in reversal of drug resistance, suggesting RAF plus HGF or MET inhibitory combination therapy as a potential therapeutic strategy (see, e.g., Hatzivassiliou et al., 2011; Straussman et al., 2012).
FGFR1 is implicated in melanoma progression, and knockdown of FGFR1 results in inhibition of melanoma growth in vivo (see, e.g., Wang et al., 1997). Fibroblast growth factor (FGF) rescues some BRAF mutant cells from treatment with PLX4032 (see, e.g., Wilson et al., 2012) and FGFR1 inhibition is synergistic with multikinase/BRAF inhibitor sorafenib and specific BRAF inhibitor RG7204 (see, e.g., Metzner et al., 2012). These findings suggest that inhibition of RTKs such as EGFR, PDGFR-β, HGFR, IGF1R and FGFR, and of SFKs should target a number of resistance mechanisms to selective BRAF inhibitors and consequently be of utility in BRAF mutant tumours that become resistant to BRAF-selective inhibitors.
Cancer and RAS
RAS proteins are small-guanine nucleotide binding proteins that are downstream of growth factor, cytokine and hormone receptors. These cell surface receptors activate proteins called guanine-nucleotide exchange factors (GNEFs), which replace GDP for GTP on RAS proteins, stimulating RAS activation. Other proteins called GTPase-activating proteins (GAPs) stimulate the intrinsic GTPase activity of RAS, thereby promoting GTP hydrolysis and returning RAS to its inactive GDP-bound state. Activated RAS binds to several effector proteins, including phosphoinositide 3-kinase (PI3K), the RAF family of protein kinases, and the Ral guanine-nucleotide exchange factor. These effectors in turn regulate the activity of the signalling pathways that control cell proliferation, senescence, survival, and differentiation. There are three RAS genes in mammals called HRAS, KRAS and NRAS and they serve overlapping but non-conserved functions.
RAS proteins are also important in cancer. 20-30% of human tumours harbour somatic gain-of-function mutations in one of the RAS genes. Most commonly these involve the codons for glycine 12 (G12), glycine 13 (G13) and glutamine 61 (Q61) and these mutations impair, through different mechanisms, the GAP-stimulated intrinsic GTPase activity of RAS, trapping it in the active GTP-bound state and allowing it to promote tumourigenesis. See, e.g., Downward et al., 2003; Young et al., 2009; Bos et al., 1989.
TABLE 2Frequency of RAS Mutations in Different Types of CancersTumour TypeFrequencyCitationPancreas90%Thyroid (Undifferentiated60%papillary)Thyroid (Follicular)55%Colorectal45%Seminoma45%Lung adenocarcinoma35%(non-small-cell)Liver30%Haematologic malignancies:Ward et al., 2012Acute myelogenous leukemia16%(AML)Juvenile myelomonocytic25%leukemia (JMML)Chronic myelomonocytic30%leukemia (JMML)Myelodisplastic 6%syndrome (MDS)Acute lymphoblastic14%leukemia (ALL)Multiple Myeloma (MM)26%Burkitt's lymphoma10%Hodgkin's lymphoma16%Malignant Melanoma20%Bladder Transitional12%Fernandes-Medarde, 2011Cell carcinomaKidney10%Epithelial ovarian cancers11%www.mycancergenome.orgLow grade serous (Type I)33%Mucinous (Type I)50-75%   Endometrial cancers0-46% Mammas et al., 2005Cervical cancer0-61% Mammas et al., 2005Biliary tract adenocarcinoma35%Fernandes-Medarde, 2011Soft tissue sarcoma*Fernandes-Medarde, 2011Angiosarcoma49%Leiomyosarcoma 8%Rhabdomyosarcoma11%Myxoma11%Malignant fibrous16%histiocytoma*The most frequently mutated RAS quoted (KRAS or NRAS or HRAS).
Other cancers have less frequent mutations of the RAS family genes, but their mutation is predictive of prognosis, for example neuroblastoma (8% NRAS mutation), stomach adenocarcinoma (6% KRAS mutant).
RAS and RAF
Active RAS proteins activate several downstream effectors, including the proteins of the RAF family. There are three RAF proteins, ARAF, BRAF and CRAF. Activated RAF phosphorylates and activates a second protein kinase called MEK, which then phosphorylates and activates a third protein kinase called ERK. ERK phosphorylates a multitude of cytosolic and nuclear substrates, thereby regulating cell processes such as proliferation, survival, differentiation and senescence.
Notably, however, in cancer cells, oncogenic RAS does not signal through BRAF, but instead signals exclusively through CRAF to activate MEK.
In the vast majority of cancers, BRAF and RAS mutations are mutually exclusive. This provides genetic evidence to suggest that these proteins are on the same pathway and that they drive the same processes in cancer cells. However, there are clear differences between oncogenic BRAF and oncogenic RAS functions in cancer cells. First, RAS activates several pathways, whereas BRAF is only known to activate the MEK/ERK pathway. As a consequence, BRAF mutant cells are more dependent on MEK/ERK signalling and so are considerably more sensitive to BRAF or MEK inhibitors than cell in which RAS is mutated. See, e.g., Garnett et al., 2004; Wellbrock et al., 2004; Gray-Schopfer et al., 2007; Solit et al., 2008.
Apart from mutations, signalling proteins in the MAPK cascade are overexpressed in a number of malignancies. For example, HRAS and NRAS are overexpressed in cervical cancers. RAS mutations are rare in adrenocortical carcinoma, but the collective population of tumours with mutations in RAS, BRAF and EGFR show increased signalling through the pathway and can be a target for MAPK pathway inhibitors (see, e.g., Kotoula et al., 2009). Low grade ovarian cancers and peritoneal cancer respond to blockage of the MAPK pathway with MEK inhibitor selumetinib independent of the RAS/RAF mutation status. In uveal melanoma, the MAPK pathway is activated through the mutation of GNAQ which accounts for 50% of uveal melanomas (see, e.g., Gaudi et al., 2011). cRAF is overexpressed in a variety of primary human cancers, such as lung, liver, prostate, primitive neurodermal tumours, head and neck squamous cell carcinoma (see, e.g., Damodar Reddy et al., 2001; Hwang et al., 2004; Mukterjee et al., 2005; Schreck et al., 2006; Riva et al., 1995). The MAPK pathway is activated in 74% of acute myeloid leukemia patients samples (see; e.g., Milella et al., 2001). In neurofibromatosis type 1, loss of NF1 tumour suppressor gene leads to hyperactivated RAS signalling, and deregulated Ras/ERK signalling which is critical for the growth of NF1 peripheral nerve tumours (see, e.g., Jessen et al., 2013). A panRAF inhibitor elicit an effective blockade of the MAPK pathway for BRAF mutant tumours and for RAS mutant tumours and has broad application for cancers with deregulation of the MAPK signalling pathway.
Cancers with activating mutations of RAS, RAF and EGFR or over expression of RAS, RAF and EGFR including any of the isoforms thereof, may be therefore particularly sensitive to panRAF (e.g., CRAF and BRAF) inhibition. Cancers with other abnormalities leading to an upregulated RAF-MEK-ERK pathway signal may also be particularly sensitive to treatment with inhibitors of panRAF (e.g., CRAF an BRAF) activity. Examples of such abnormalities include constitutive activation of a growth factor receptor, overexpression of one or more growth factor receptors; overexpression of one or more growth factors; KSR-mediated pathway activation; and BRAF or CRAF gene fusions.
MAPK Pathway in Other Diseases
The RAF-MEK-ERK pathway functions downstream of many receptors and stimuli indicating a broad role in regulation of cell function. For this reason, inhibitors of RAF may find utility in other disease conditions that are associated with up-regulation of signalling via this pathway. The RAF-MEK-ERK pathway is also an important component of the normal response of non-transformed cells to growth factor action. Therefore, inhibitors of RAF may be of use in diseases where there is inappropriate or excessive proliferation of normal tissues. These include, for example, glomerulonephritis and psoriasis.
The function of inflammatory cells is controlled by many factors, the effects of which are mediated by different signal transduction pathways. Although some key pro-inflammatory functions are mediated by p38 Map kinase (e.g., TNF release), others are mediated by other pathways. The RAF-MEK-ERK pathway, in particular, is an important activating and proliferative signal in many inflammatory cells. B and T lymphocytes, in particular, require activation of the RAF-MEK-ERK pathway for clonal expansion and generation of effector populations (see, e.g., Cantrell, 2003; Genot et al., 2000). The cellular signalling pathway of which RAF is a part has been implicated in inflammatory disorders characterized by T-cell proliferation (T-cell activation and growth), such as tissue graft rejection, endotoxin shock, and glomerular nephritis.
Activation of the MAPK/ERK signalling has been demonstrated in many models of disease models, and inhibition of the pathway, using for example MEK inhibitors, have been shown to be potentially beneficial in these various diseases such as:                Pain: Evidence of Efficacy in Pain Models: MEK pathway is upregulated in dorsal horn neurons in persistent pain (see, e.g., Ji et al., 2002; Song et al., 2005; Ma et al., 2005; Karim et al., 2006); Mek inhibitors in neuropathic pain (see, e.g., Dixon et al., 2001).        Stroke: Evidence of Efficacy in Stroke Models Significant Neuroprotection against Ischemic Brain Injury by Inhibition of the MEK (see, e.g., Wang et al., 2003; Wang et al., 2004; Maddahi et al., 2010).        Diabetes: Evidence In Diabetic Complications (see, e.g., Fujita et al., 2004).        Inflammation: Evidence of Efficacy in Inflammation Models (see, e.g., Jaffee et al., 2000; Thalhamer et al., 2008; Geppert et al., 1994).        Arthritis: Evidence of efficacy in experimental osteoarthritis (see, e.g., Pelletier et al., 2003); model of rheumatoid arthritis (see, e.g., Chun et al., 2002; Dudley et al., 2000); reviewed in Thalhamer et al., 2008.        Heart remodelling, for example, in metabolic syndrome (see, e.g., Asrih et al., 2013).        Organ injury, for example, in cisplatin-induced renal injury (see, e.g., Jo et al., 2005).        Haemoglobinopathies: sickle-cell disease, β-thalassemia, haemoglobin H disease (see, e.g., Zennadi et al., 2012).        Asthma (see, e.g., Bridges et al., 2000).        Transplant rejection (see, e.g., Gilbertsen et al., 2000).        Septic shock (see, e.g., Geppert et al., 1994).        Viral infection, for example hepatitis B (see, e.g., Benn et al., 1994), hepatitis C (see, e.g., Zhang et al., 2012), human immunodeficiency virus (HIV) (see, e.g., Yang et al., 1999), Epstein-Barr virus (EBV) (see, e.g., Fukuda et al., 2007), HPV (see, e.g., Payne et al., 2001), human herpesvirus-8 (HHV) associated with Kaposi sarcoma (see, e.g., Akula et al., 2004), human cytomegalovirus (see, e.g., Johnson et al., 2001), Coxsackievirus B3 (see, e.g., Luo et al., 2002), Boma virus (see, e.g., Planz et al., 2001), influenza virus (see, e.g., Pleschka et al., 2001).        chronic infections and autoimmune diseases, for example, by Inhibiting regulatory T-cells activity (see, e.g., Kietil et al., 2013).        Atherosclerosis (see, e.g., Miura et al., 2004).        Restenosis (see, e.g., Graf et al., 1997).        Cardiomyopathy (see, e.g., Lorenz et al., 2009).        Cardiac ischemia reperfusion injury (see, e.g., Zouki et al., 2000).        Psoriasis (see, e.g., Haase et al., 2001).        Alzheimer's disease (see, e.g., Mei et al., 2006) and other induced neurological disorders such as HTLV-I-associated myelopathy/tropical spastic parasite or neurodegenerative diseases such as Parkinson's disease or Amyloid Lateral Sclerosis via CD44 splice-variants modulation (see, e.g., Pinner et al., 2009).        Chronic obstructive pulmonary disorder (see, e.g., Mercer et al., 2006).        Inflammatory bowel disease (see, e.g., Lowenberg et al., 2005).        Fibrogenetic diseases, such as cystic fibrosis (see, e.g., Li et al., 1998), liver fibrosis, for example, liver cirrhosis (see, e.g., Davies et al., 1996).        Hereditary RAS mutations lead to a group of diseases named collectively as rasopathy. Targeting the MAPK pathway in these diseases has been proposed as a therapeutic approach in these type of diseases such as Noonan Syndrome (see, e.g., Gu et al., 2013), Cardiofaciocutaneous Syndrome (see, e.g., Anastasaki et al., 2012) and capillary malformations (see, e.g., Vikkula et al., 2004).RTKs        
Receptor tyrosine kinases (RTKs) are important in the transmission of biochemical signals across the plasma membrane of cells. These transmembrane molecules characteristically consist of an extracellular ligand-binding domain connected through a segment in the plasma membrane to an intracellular tyrosine kinase domain. Binding of ligand to the receptor results in stimulation of the receptor-associated tyrosine kinase activity that leads to phosphorylation of tyrosine residues on both the receptor and other intracellular proteins, leading to a variety of cellular responses. To date, at least nineteen distinct RTK subfamilies, defined by amino acid sequence homology, have been identified.
FGFR
The fibroblast growth factor (FGF) family of signaling polypeptides regulates a diverse array of physiologic functions including mitogenesis, wound healing, cell differentiation and angiogenesis, and development. Both normal and malignant cell growth as well as proliferation are affected by changes in local concentration of these extracellular signalling molecules, which act as autocrine as well as paracrine factors. Autocrine FGF signalling may be particularly important in the progression of steroid hormone-dependent cancers and to a hormone independentstate (see, e.g., Powers et al., 2000).
FGFs and their receptors are expressed at increased levels in several tissues and cell lines. and overexpression is believed to contribute to the malignant phenotype. Furthermore, a number of oncogenes are homologues of genes encoding growth factor receptors, and there is a potential for aberrant activation of FGF-dependent signaling in human pancreatic cancer (see, e.g., Ozawa et al., 2001).
The two prototypic members are acidic fibroblast growth factor (aFGF or FGF1) and basic fibroblast growth factors (bFGF or FGF2), and to date, at least twenty distinct FGF family members have been identified. The cellular response to FGFs is transmitted via four types of high affinity transmembrane tyrosine-kinase fibroblast growth factor receptors numbered 1 to 4 (FGFR-1 to FGFR-4). Upon ligand binding, the receptors dimerize and auto- or trans-phosphorylate specific cytoplasmic tyrosine residues to transmit an intracellular signal that ultimately reaches nuclear transcription factor effectors.
Disruption of the FGFR-1 (FGFRA) pathway should affect tumour cell proliferation since this kinase is activated in many tumour types in addition to proliferating endothelial cells. The overexpression and activation of FGFR-1 in tumour-associated vasculature has suggested a role for these molecules in tumour angiogenesis.
FGFR-2 has high affinity for the acidic and/or basic fibroblast growth factors, as well as the keratinocyte growth factor ligands. FGFR-2 also propagates the potent osteogenic effects of FGFs during osteoblast growth and differentiation. Mutations in FGFR-2, leading to complex functional alterations, were shown to induce abnormal ossification of cranial sutures (craniosynostosis), implying a major role of FGFR signaling in intramembranous bone formation. For example, in Apert (AP) syndrome, characterized by premature cranial suture ossification, most cases are associated with point mutations engendering gain-of-function in FGFR 2 (ooc, e.g., Lemonnier et al., 2001).
Lemonnier et al., 2001, “Role of N-cadherin and protein kinase C in osteoblast gene activation induced by the S252W fibroblast growth factor receptor 2 mutation in Apert craniosynostosis”, J. Bone Miner, Res., Vol. 16, pp. 832-845.
Several severe abnormalities in human skeletal development, including Apert, Crouzon, Jackson-Weiss, Beare-Stevenson cutis gyrata, and Pfeiffer syndromes are associated with the occurrence of mutations in FGFR-2. Most, if not all, cases of Pfeiffer Syndrome (PS) are also caused by de novo mutation of the FGFR-2 gene (see, e.g., Meyers et al., 1996; Plomp et al., 1998), and it was recently shown that mutations in FGFR-2 break one of the cardinal rules governing ligand specificity. Namely, two mutant splice forms of fibroblast growth factor receptor, FGFR2c and FGFR2b, have acquired the ability to bind to and be activated by atypical FGF ligands. This loss of ligand specificity leads to aberrant signalling and suggests that the severe phenotypes of these disease syndromes result from ectopic ligand-dependent activation of FGFR-2 (see, e.g., Yu et al., 2000).
Activating mutations of the FGFR-3 receptor tyrosine kinase such as chromosomal translocations or point mutations produce deregulated, constitutively active, FGFR-3 receptors which have been involved in multiple myeloma and in bladder and cervix carcinomas (see, e.g., Powers et al., 2000). Accordingly, FGFR-3 inhibition would be useful in the treatment of multiple myeloma, bladder, and cervix carcinomas.
Angiogenesis
Chronic proliferative diseases are often accompanied by profound angiogenesis, which can contribute to or maintain an inflammatory and/or proliferative state, or which leads to tissue destruction through the invasive proliferation of blood vessels. See, e.g., Folkman, 1995; Folkman, 1997; Folkman et al., 1992.
Angiogenesis is generally used to describe the development of new or replacement blood vessels, or neovascularisation. It is a necessary and physiological normal process by which the vasculature is established in the embryo. Angiogenesis does not occur, in general, in most normal adult tissues, exceptions being sites of ovulation, menses, and wound healing. Many diseases, however, are characterized by persistent and unregulated angiogenesis. For instance, in arthritis, new capillary blood vessels invade the joint and destroy cartilage (see, e.g., Colville-Nash and Scott, 1992). In diabetes (and in many different eye diseases), new vessels invade the macula or retina or other ocular structures, and may cause blindness (see, e.g., Alon et al., 1995). The process of atherosclerosis has been linked to angiogenesis (see, e.g., Kahlon et al., 1992). Tumour growth and metastasis have been found to be angiogenesis-dependent (see, e.g., Folkman, 1992; Denekamp, 1993; Fidler and Ellis, 1994).
The recognition of the involvement of angiogenesis in major diseases has been accompanied by research to identify and develop inhibitors of angiogenesis. These inhibitors are generally classified in response to discrete targets in the angiogenesis cascade, such as activation of endothelial cells by an angiogenic signal; synthesis and release of degradative enzymes; endothelial cell migration; proliferation of endothelial cells; and formation of capillary tubules. Therefore, angiogenesis occurs in many stages and attempts are underway to discover and develop compounds that work to block angiogenesis at these various stages.
There are many publications that teach that inhibitors of angiogenesis, working by diverse mechanisms, are beneficial in diseases such as cancer and metastasis (see, e.g., O'Reilly et al., 1994; Ingber et al., 1990), ocular diseases (see, e.g., Friedlander et al., 1995), arthritis (see, e.g., Peacock et al., 1992; Peacock et al., 1995), and hemangioma (see, e.g., Taraboletti et al., 1995).
VEGFR
Vascular endothelial growth factor (VEGF), a polypeptide, is mitogenic for endothelial cells in vitro and stimulates angiogenic responses in vivo. VEGF has also been linked to inappropriate angiogenesis (see, e.g., Pinedo et al., 2000). VEGFR(s) are receptor tyrosine kinases (RTKs). RTKs catalyze the phosphorylation of specific tyrosyl residues in proteins involved in the regulation of cell growth and differentiation (see, e.g., Wilks et al., 1990; Courtneidge et al., 1993; Cooper et al., 1994; Paulson et al., 1995; Chan et al., 1996).
Three RTK receptors for VEGF have been identified: VEGFR-1 (Flt-1), VEGFR-2 (Flk-1 or KDR), and VEGFR-3 (Fit-4). These receptors are involved in angiogenesis and participate in signal transduction (see, e.g., Mustonen et al., 1995).
Of particular interest is VEGFR-2 (KDR), which is a transmembrane receptor RTK expressed primarily in endothelial cells. Activation of VEGFR-2 by VEGF is a critical step in the signal transduction pathway that initiates tumour angiogenesis. VEGF expression may be constitutive to tumour cells and can also be up-regulated in response to certain stimuli. One such stimuli is hypoxia, where VEGF expression is upregulated in both tumour and associated host tissues. The VEGF ligand activates VEGFR-2 by binding with its extracellular VEGF binding site. This leads to receptor dimerization of VEGFRs and auto-phosphorylation of tyrosine residues at the intracellular kinase domain of VEGFR-2. The kinase domain operates to transfer a phosphate from ATP to the tyrosine residues, thus providing binding sites for signalling proteins downstream of VEGFR-2 leading ultimately to initiation of angiogenesis (see, e.g., McMahon et al., 2000).
Inhibition at the kinase domain binding site of VEGFR-2 would block phosphorylation of tyrosine residues and serve to disrupt initiation of angiogenesis.
VEGFR-2 (and VEGFR-3) are primarily localized to the tumour vasculature (blood and/or lymphatic) supporting the majority of solid cancers, and is significantly upregulated. The primary clinical mechanism of action of VEGF signaling inhibitors is likely to be through the targeting of tumour vessels rather than tumour cells (see, e.g., Smith et al., 2010), although other mechanisms have been described. Vascular endothelial growth factor (VEGF)-targeted agents, administered either as single agents or in combination with chemotherapy, have been shown to benefit patients with advanced-stage malignancies (see, e.g., Ellis et al., 2008).
KDR plays a crucial role in other diseases, and inhibitors of KDR may find utility in these conditions.
Atherosclerosis: KDR is strongly expressed both on endothelial cells during angiogenesis and on the luminal endothelium of human atherosclerotic vessels, but not in normal arteries or veins (see, e.g., Belgore et al., 2004). The interaction between VEGF and VEGF receptor 2 (KDR, human; Flk-1, mouse) is key to pathologic angiogenesis and has been implicated in the development of atherosclerotic lesions (see, e.g., Inoue et al., 1998). Vaccination against KDR resulted in T-cell activation, suppression of neo-angiogenesis, and a marked reduction in atherosclerosis which was independent of hypercholesterolemia in both male and female mice (see, e.g., Petrovan et al., 2007).
Obesity: Formation of new vessels in fat tissues during diet-induced obesity is largely due to angiogenesis rather than de novo vasculogenesis. Anti-angiogenic treatment by blockade of VEGFR2 but not VEGFR1 may limit adipose tissue expansion (see, e.g., Tam et al., 2009).
Retinopathy and Maculopathy: Abnormal activation of the VEGF-VEGFR system is intimately involved in the progression of age-related macular degeneration (AMD). Therefore, an aptamer against VEGF-A165, a VEGF-neutralizing antibody (Fab type) and VEGF-Trap are now approved for AMD treatment (see, e.g., Masabumi et al., 2013). Bevazucimab, an anti-VEGF antibody, is used off-label in conditions such as AMD, diabetic retinopathy, and diabetic macular edema (DME) (see, e.g., Rotsos et al., 2008).
Neuropathic pain syndrome: VEGF and VEGFR2 are involved in the pathogenesis of neuropathic pain. Anti-rVEGF treatment in CCI rats may alleviate chronic neuropathic pain by decreasing the expressions of VEGFR2 and P2X2/3 receptors on DRG neurons to inhibit the transmission of neuropathic pain signaling (see, e.g., Lin et al., 2010).
Rheumatoid arthritis: PTK787/ZK222584, a receptor tyrosine kinase inhibitors with specific activity against the VEGFRs, and that exhibits strong inhibition of VEGF-R2 (KDR) and slightly weaker inhibition of VEGFR1 (Flt-1), FIk-1 (the mouse homologue of KDR), and Fit-4 (the receptor found in the lymphatic system), inhibited knee swelling by 40%, severity scores (by 51%) and global histological scores in mice with collagen-induced arthritis (see, e.g., Grosios et al., 2004)
TIE
Angiopoietin 1 (Ang1), a ligand for the endothelium-specific receptor tyrosine kinase TIE-2 is an angiogenic factor (see, e.g., Davis et al., 1996; Partanen et al., 1992; Davis et al., 1994; Davis et al., 1996; Alitalo et al., 1996; Godowski et al., 1997). The acronym TIE represents “tyrosine kinase containing g and EGF homology domains”. TIE is used to identify a class of receptor tyrosine kinases, which are exclusively expressed in vascular endothelial cells and early hemopoietic cells. Typically, TIE receptor kinases are characterized by the presence of an EGF-like domain and an immunoglobulin (IG) like domain, which consists of extracellular folding units, stabilized by intra-chain disulfide bonds (see, e.g., Partanen et al., 1999). Unlike VEGF, which functions during the early stages of vascular development, Ang1 and its receptor TIE-2 function in the later stages of vascular development, i.e., during vascular remodelling (remodelling refers to formation of a vascular lumen) and maturation (see, e.g., Yancopoulos et al., 1998; Peters et al., 1998; Suri et al., 1996).
Consequently, inhibition of TIE-2 would be expected to serve to disrupt remodelling and maturation of new vasculature initiated by angiogenesis thereby disrupting the angiogenic process.
p38
p38 is a MAPK family member of 38 kDa that is activated in response to stress and plays an important role in the immune response and cell survival and differentiation. Four p38 MAPK kinases have been described; these proteins share a high degree of homology (p38α, β, γ, and δ). p38 MAPKs can be activated by different stimuli such as growth factors, inflammatory cytokines, or a variety of environmental stresses. p38 MAPKs can in turn activate a number of downstream targets, including protein kinases, cytosolic substrates, transcription factors and chromatin remodeling factors. Strong activation of p38 MAPKs by cytokines and cellular stresses generally promotes the inhibition of cell growth and induces apoptosis (see, e.g., review in Cuadrado et al., 2010). More recently, p38α has been found to play important roles in the maintenance of homoeostasis and related pathologies. The best-known and most widely reported role of p38α in disease is related to its function in cytokine signaling and promotion of pathological inflammation. Several studies have shown how p38α can mediate a series of disease models, including rheumatoid arthritis, psoriasis, Alzheimer's disease, inflammatory bowel disease, Crohn's disease, tumourigenesis, Cardiovascular disease, and stroke. Moreover, there is evidence of a role for p38 MAPK in the development and maintenance of a number of pulmonary diseases, such as asthma, cystic fibrosis, idiopathic pulmonary fibrosis, and chronic obstructive pulmonary disease.
Thus, p38α is an interesting pharmaceutical target especially because of its important role in inflammatory diseases (see, e.g., review in Oeztuerk-Winder et al., 2012). Pyridinyl-imidazole drugs such as SB8203580 were the first p38 MAPK inhibitors to be identified that bind competitively at the ATP-binding pocket, and have been widely used to study p38 MAPK functions (see, e.g., Coulthard et al., 2009).
SRC
c-SRC belongs to the non-receptor SRC family kinases (SFKs). These proteins are involved in many cellular events such as proliferation, survival, and cell motility. Thus, hyper-activation of SRC signaling contributes to diverse aspects of tumour development. The most prominent function of c-SRC is its extensive interaction with transmembrane receptor tyrosine kinases (RTKs) at the cell membrane via its SH2 and SH3 domains. c-SRC interacts with many RTKs including epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), platelet-derived growth factor receptor (PDGFR), insulin-like growth factor-1 receptor (IGF-1R) and c-Met/hepatocyte growth factor receptor (HGFR). Through these interactions, c-SRC integrates and regulates RTK signaling and directly transduces survival signals to downstream effectors such as phosphoinositide 3-kinases (PI3Ks), Akt, and signal transducer and activator of transcription 3 (STAT3) (see, e.g., Zhang et al., 2012). Other membrane receptors such as integrins can also activate c-SRC thus triggering a signal cascade that regulates cell migration adhesion and invasion. c-Src activation through the interaction with p120 catenin promotes dissociation of cell-cell adherens junctions thus enhancing cell motility. c-SRC directly phosphorylates the focal adhesion kinase (FAK) stabilizing focal adhesion complexes, which consist of FAK, paxillin, RhoA, and other components, and enhances cell adhesion to the extracellular matrix. Furthermore, c-SRC plays an important role in regulating the tumour microenvironment. c-SRC activation in hypoxia promotes angiogenesis through stimulation of the expression of vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMPs) and interleukin-8 (IL-8) (see, e.g., Yeatman et al., 2004).
Targeting SFKs is a well established therapeutic approach for many types of cancer. Dasatinib is an orally available small-molecule multi-kinase inhibitor that potently inhibits SRC-family kinases (SRC, LCK, YES, FYN), but also BCR-ABL, c-KIT, PDGFR-α and β, and ephrin receptor kinase (see, e.g., Lindauer et al., 2010). More recent studies have reported that Src is also involved in the inflammation-related signaling pathway. Many studies have shown that c-SRC plays a critical role in macrophage-mediated inflammatory responses. Importantly, a variety of inflammatory diseases is closely related to macrophage activation; therefore, c-SRC inhibition may represent a useful therapeutic strategy for macrophage-mediated diseases (see, e.g., Byeon et al., 2012).
Lck
Lck (lymphocyte specific kinase) is a kinase of the SFKs that is critical for T-cell activation, and its activity is induced by the T-cell receptor (TCR). TCR signals initiated by Lck lead to gene regulation events resulting in cytokine release, proliferation and survival of antigen specific T-cells thereby amplifying specific immune responses. Inhibition of Lck is expected to offer a new therapeutic approach for the treatment of T-cell-mediated autoimmune and inflammatory disorders and/or organ transplant rejection (see, e.g., Martin et al., 2010).
Known Compounds
Niculescu-Duvaz et al., 2006, describes certain imidazo[4,5-b]pyridin-2-one and oxazolo[4,5-b]pyridin-2-one compounds which, inter alia, inhibit RAF (e.g., BRAF) activity, and which are useful in the treatment of proliferative disorders such as cancer. A number of compounds shown therein have a 5-(tert-butyl)-2-(phenyl)-pyrazol-3-yl group or a 5-(tert-butyl)-2-(pyridyl)-pyrazol-3-yl group. However, in every case, the phenyl and pyridyl group is unsubstituted, para-substituted, or ortho,para-disubstituted; in none of the compounds is it meta-substituted. The following compounds are shown:
StructureCitationCJS 3247 CJS 3600 CJS 3608 CJS 3609 CJS 3614 CJS 3615 CJS 3617
Niculescu-Duvaz et al., 2007, describes certain imidazo[4,5-b]pyridin-2-one and oxazolo[4,5-b]pyridin-2-one compounds which, inter alia, inhibit RAF (e.g., BRAF) activity, and which are useful in the treatment of proliferative disorders such as cancer. A number of compounds shown therein have a 5-(tert-butyl)-2-(phenyl)-pyrazol-3-yl group. However, in every case, the phenyl group is unsubstituted or para-substituted; in none of the compounds is it meta-substituted. The following compounds are shown:
StructureCitationCJS 3683 CJS 3741 CJS 3742
Springer et al., 2009, describes certain pyrido[2,3-b]pyrazin-8-substituted compounds which, inter alia, inhibit RAF (e.g., BRAF) activity, and which are useful in the treatment of proliferative disorders such as cancer. A number of compounds shown therein have a 5-(tert-butyl)-2-(phenyl)-pyrazol-3-yl group or a 5-(tert-butyl)-2-(pyridyl)-pyrazol-3-yl group. However, in every case, the phenyl and pyridyl group is unsubstituted or para-substituted; in none of the compounds is it meta-substituted. The following compounds are shown:
StructureCitationCompound AA-018 Compound AA-019 Compound AA-062 Compound AA-084
Niculescu-Duvaz et al., 2009, describes certain aryl-quinolinyl compounds which, inter alia, inhibit RAF (e.g., BRAF) activity, and which are useful in the treatment of proliferative disorders such as cancer. A number of compounds shown therein have a 5-(tert-butyl)-2-(phenyl)-pyrazol-3-yl group. However, in every case, the phenyl group is unsubstituted or para-substituted; in none of the compounds is it mete-substituted. The following compounds are shown:
StructureCitationAA-005 AA-006 BB-007 BB-008
Springer et al., 2011, describes certain 1-(5-tert-buty-2-phenyl-2H-pyrazol-3-yl)-3-[2-fluoro-4-(1-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy)-phenyl]urea compounds which, inter alia, inhibit RAF (e.g., BRAF) activity, and which are useful in the treatment of proliferative disorders such as cancer. The following compound is shown:
StructureCitationCompound AA-04
Murray et al., 2011, describes certain compounds for use in the treatment of an inflammatory disease or a respiratory disorder. A few of the compounds shown therein have a 5-(tert-butyl)-2-(phenyl)-pyrazol-3-yl group or a 5-(tert-butyl)-2-(pyridyl)-pyrazol-3-yl group. However, in every case, the phenyl and pyridyl group is unsubstituted, para-substituted, or meta, para-substituted; in none of the compounds is it meta-substituted, para-unsubstituted. The following compounds are shown:
StructureCitationExample 18 (page 58) Example 32 (page 63)
A number of compounds having a 5-(tert-butyl)-2-(3-fluoro-phenyl)-pyrazol-3-yl group are known, including the following:
StructureCitationFuruta et al.,  2012 Flynn et al.,  2008 Smith et al.,  2007 Cantin et al., 2007